induction of high frequency somatic embryogenesis and...

Indian Journal of Experimental Biology

Vol. 56, March 2018, pp. 180-193

Induction of high frequency somatic embryogenesis and analysis of developmental

stagewise expression of SERK1 gene during somatic embryogenesis in

cultures of Vigna radiata (L.) R.Wilczek

Vajravel Sindhujaa1, Muniraj Gnanaraj

1, Maluventhen Viji

2, Thirupathi Karuppanapandian

3 & Kumariah Manoharan

1*

1Department of Plant Morphology and Algology, School of Biological Sciences, Madurai Kamaraj University,

Madurai-625 021, Tami Nadu, India 2Department of Botany, Thiagarajar College, Madurai 625 009, Tami Nadu, India

3Department of Experimental Biology, Faculty of Science, Masaryk University, Brno-625 00, Czech Republic

Received 23 June 2015; revised 13 May 2017

Vigna radiata (L.) R.Wilczek (Fabaceae), commonly called Green gram or Mung bean, is an important legume with

potential nutritional, medicinal and health benefits cultivated widespread throughout the rain-fed areas of arid and semi-arid

tropics and subtropics. Being an affordable source of carbohydrate, vitamins, minerals and phytonutrients besides protein,

Green gram finds demand for its nutrient digestibility, food processing properties and bioavailability. Though India ranks

top in world mung bean production (>50%), it is unable to meet the local demand. Biotic and abiotic stresses restrict mung

bean yield considerably and researchers have been working on resistant varieties to overcome these challenges. In this study,

towards improving yield, an effective protocol for attaining high frequency somatic embryogenesis (SE) in green gram has

been proposed. Type of explants and age of source seedlings for obtaining explants were found to influence the formation of

embryogenic calli. Various combinations and concentrations of 2,4-dichlorophenoxyacetic acid and indole-3-acetic acid

with kinetin were optimized for developing embryogenic calli. Embryogenic calli when exposed to osmotic stress created by

D-mannitol and sorbitol and dehydration stress imposed by polyethylene glycol were found to produce somatic embryos.

Calli incubated for 6 h in specified hormone free nutrient medium supplemented with 4% polyethylene glycol was optimal

for induction of high frequency SE. Subsequent to stress incubation, the cultures formed only early stage somatic embryos.

Supplementation of proline was found essential for the maturation of somatic embryos. Cotyledonary stage somatic embryos

were converted into plantlets and subsequently established in garden soil. Semi-quantitative Reverse Transcription-PCR

based transcript level analysis of SERK1 gene expression was carried out during different developmental stages of somatic

embryogenesis. Expression of SERK1 was specifically associated with the embryogenic calli and calli enriched with

globular stage somatic embryos.

Keywords: Embryogenic competence, Green gram, Mung bean, Proline, Polyethylene glycol

Vigna radiata (L.) R.Wilczek is an important nitrogen

fixing nutritionally rich legume, commonly called

Mung bean or Green gram, cultivated mostly in rain-

fed areas of arid and semi-arid tropics and subtropics1-

3. Besides protein, it is an affordable source of

phytonutrients including carbohydrate, vitamins,

minerals, dietary fibre with antioxidant, antimicrobial,

anti-inflammatory, antihypertensive, antitumor and

antidiabetic potentials3,4

. In India, green gram has

been a traditional diet since ancient time for its

nutrient digestibility, medicinal values and

bioavailability3. Recent study has shown that the

sprouts of green gram are useful in treating non-

alcoholic fatty liver disease and alterations related to

metabolic syndrome5.

Though India leads in world mung bean acerage

(30.41 lakh tonnes/ha) and production (14.24 lakh

tonnes), due to high local consumption it is unble to

meet the demand completely2. Moreover, despite

large area of cultivation, the total productivity is low

(468 kg/ha) because of several biotic and abiotic

stress factors that affect plant growth and

development2,6,7

. Apart from insect pests such as hairy

caterpillar, jassids, galerucide beetle and whitefly

other biotic factors that affect crop productivity

include viral diseases, such as yellow mosaic disease

(mungbean yellow mosaic virus), leaf crinkle disease

(urdbean leaf crinkle virus) and leaf curl/necrosis

———————

*Correspondence:

E-mail: [email protected]

Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; EC,

Embryogenic calls; IAA, Indole-3-acetic acid; Kn, kinetin; PEG,

Polyethylene glycol; SE, Somatic Embryogenesis; SFIM, stress

factor incubation medium; WM, washing medium

SINDHUJAA et al.: SOMATIC EMBRYOGENESIS IN GREEN GRAM

181

disease (groundnut bud necrosis virus), fungal diseases,

such as, powdery mildew (Erysiphe polygoni),

anthracnose (Colletotrichum lindemuthianum), leaf

spot (Cercospora canescens), rust (Uromyces phaseoli)

and dry root-rot (Rhizoctonia bataticola), and bacterial

disease, viz., leaf blight (Xanthomonas phaseoli) and

Macrophomina blight (Macrophomina phaseoli)2,6,7

.

The abiotic stress factors affecting the greengram

productivity include drought, salinity and heavy

metal toxicity8.

Agronomic traits, such as tolerance to extreme temperatures, drought, avoidance of pre-harvest sprouting during rainy season by evolving short duration genotypes and nutritional enhancement in grain quality involving higher protein content and amino acid composition with reference to essential amino acids of seed proteins have been identified to be the desirable traits for improving the crop productivity of this species

9. However, conventional

crop improvement is limited due to narrow genetic base and sexual incompatibility with wild relatives. Hence, genetic engineering methodologies need to be employed for augmenting the agronomic traits

10. An

efficient protocol for high frequency plantlet regeneration is a prerequisite for application of genetic transformation methodologies

11. Greengram

in vitro is found to be recalcitrant, especially for high frequency plantlet regeneration

12,13. Somatic

embryogenesis (SE) is ideal for high frequency plantlet regeneration that could be used in genetic transformation studies. As compared to plantlet regeneration via organogenesis, development of somatic embryo derived plantlets offer an attractive advantage for raising genetically homogeneous plantlets without the formation of chimeras for transgenic studies

11.

Inductive conditions for SE in greengram have

been reported, indicating choice of explants and type

and concentration of phytohormones and growth

factors13,14

. Abiotic stress factors, such as sorbitol,

D-mannitol and polyethylene glycol (PEG) have

been employed in embryogenic callus cultures in

order to induce either formation or maturation of

somatic embryos in diverse plants, such as, carrot

(Daucus carota)15

, alfalfa (Medicago sativa)16

,

thale cress (Arabidopsis thaliana)17

and white spruce

(Picea glauca)18

.

Previous study in our laboratory has led to

establishment of abiotic stress factor induced SE in

cultures of pigeonpea19

. In this study, we attempted

the following: (i) to suggest a high throughput plantlet

regeneration system via SE by working out a widely

applicable experimental approach for optimization of

inductive conditions for SE in the recalcitrant species;

and (ii) to characterize the presence of SERK1 gene in

embryogenic callus cultures and the timing of its

expression during SE in cultures of greengram.

Materials and Methods Seeds of greengram [Vigna radiata (L.) Wilczek]

cv. CO-6 were obtained from Tamil Nadu Agriculture

University, Coimbatore, India. Seeds were washed

thrice with sterile distilled water, followed by

treatment with 0.1% HgCl2 for 4 min and

subsequently rinsed 5 times with sterile distilled

water. Seeds were germinated aseptically on

semisolid 0.7% agar-water medium. Seedlings,

cultures and plantlets derived from somatic embryos

were maintained at 25±1°C under white fluorescent

light (15 µmol m-2

s-1

) with a light-dark cycle of

16/8 h and RH of 80%.

Preparation of explants

Three day old sprouts and 5 day old seedlings were

placed on separate Petri dishes lined with two layers

of moist sterile Whatman No.1 paper for dissecting

the explants. Embryo axis from the sprouts with cut

ends on both sides, cotyledonary node and leaf from

the seedlings were excised and cultured. Leaves were

excised into ca. 4 mm2 pieces and cultured with their

abaxial surface in contact with the medium. Twelve

explants per culture vessel (Borosil 250 ml

Erlenmeyer flask) were inoculated.

Determination of morphometric parameters in relation to

callusing response in cultures

Culture responses related to % callusing response,

colour and texture of the calli, chlorophyll content of

the calli, embryogenic/non-embryogenic nature of

calli, % embryogenic cells and amount of calli

produced per explant were routinely determined in

28 day old primary callus cultures.

Nutrient media, experimental solutions and conditions

Murashige and Skoog (MS)20

medium was used

as the basal medium along with specified

phytohormones, osmolytes and amino acids. The pH

of the media was adjusted to 5.6 with 0.1 M KOH

before autoclaving at 15 psi for 15 min. The different

nutrient media employed in the present study are

listed in Table 1. The in vitro developed somatic

embryo derived plantlets were grown in a mixture of

sterilized vermiculite, sand and soil (2:1:1) in paper

INDIAN J EXP BIOL, MARCH 2018

182

cups and irrigated with Hoagland’s nutrient medium.

The plantlets transferred to paper cups were covered

with polythene bags during hardening. When the

plantlets had shown signs of acclimatization, the

polythene covers were removed. Four week old

hardened plantlets were found ideal for transfer to

the field.

Incubation in osmolytes

Twenty eight day old embryogenic primary calli derived from the embryo axis explants were

incubated in individual osmolyte containing (PEG/mannitol/sorbitol) hormone free stress factor

incubation medium (SFIM) for specified durations of

0, 3, 6 and 9 hours. Stress incubation of calli was carried out in an orbital shaker (New Brunswick,

USA) at 100 rpm. Stress factor incubated calli (ca. 6 g fw) in ca. 120 mL SFIM was pelleted by

centrifugation at 500×g for 3 min. Subsequently, the callus cells were washed 3X by centrifugation by

using the washing medium (WM). The supernatant

was withdrawn by using Pasteur pipette. The pelleted callus tissue was subsequently cultured on the

semisolid SEM with ca. 100 mg inocula in Borosil 250 mL Erlenmeyer flasks.

Chronology of the cultures

Primary callus cultures initiated from different

explant types were employed for working out the

conditions for developing embryogenic calli. The % callusing response was scored at the time of

emergence of callus from the different explants types. Colour and texture of the calli and other parameters

related to callusing response were determined on the 28

th day of the primary cultures. The primary callus

cultures after 28 days of growth were employed for

stress factor incubation to induce SE. Evaluation for the occurrence of different stages of somatic embryos

was done on 28th day subsequent to stress factor

incubation in the I subcultured calli. The second

subculture involved the supplementation of various

amino acids to evaluate the effect on the maturation of somatic embryos. The matured somatic embryos

obtained after 28 days in II subculture were transferred to plantlet regeneration medium. The

developed plantlets were subsequently transferred to vermiculite:sand:soil mix.

Microscopic observations of embryogenic calli and evaluation

of frequency of somatic embryo formation

The following calli were evaluated microscopically

for scoring the frequency of embryogenic cells in

Table 1 — Nutrient media employed for the formation of embryogenic calli, induction of somatic embryogenesis, development of

matured somatic embryos and regeneration of plantlets in cultures of green gram

Medium composition Experimental purpose and medium abbreviation

Semisolid MS + 2,4-D 9/18/27/36/45µM (solified with 0.7 % agar) Initiating callus cultures from different explants / developing

embryogenic calli; Callusing Medium (CM)

Semisolid MS + 2,4-D 18 µM + kn 2.3/4.6/6.9/9.2 µM Callusing from embryo axis and leaf explants/ developing

embryogenic calli; Callusing Medium (CM-EA)

Semisolid MS + 2,4-D 27 µM + kn 2.3/4.6/6.9/9.2 µM Callusing from cotyledonary node explants/ developing

embryogenic calli; Callusing Medium (CM-CN)

Semisolid MS + IAA 18 µM Callusing from embryo axis and leaf explants/ developing

embryogenic calli; callusing medium (CM-IAA-EA/L)

Semisolid MS + IAA 27 µM Callusing from cotyledonary node explants/ developing

embryogenic calli; callusing medium (CM- IAA-CN)

Liquid, hormone free MS + mannitol 0.6/0.7/0.8 M Stress incubation medium for embryo axis derived calli

(SIM-mannitol)

Liquid, hormone free MS + sorbitol 0.6/0.7/0.8 M Stress incubation medium for embryo axis derived calli (SIM-

sorbitol)

Liquid, hormone free MS + PEG 3/4/5 % (w/v) Stress incubation medium for embryo axis derived calli (SIM-PEG)

Liquid, MS + 2,4-D 18 µM Washing the stress factor incubated calli; washing medium (WM)

Semisolid, MS + 2,4-D 18 µM Development of somatic embryos; somatic embryogenesis medium

(SEM)

Semisolid MS + 2,4-D 18 µM +tryptophan 50, 100, 150, 200 mM Maturation of somatic embryos; somatic embryo maturation

medium (SEMM-Try)

Semisolid MS + 2,4-D 18 µM + glutamine 50, 100, 150, 200 mM Maturation of somatic embryos; somatic embryo maturation

medium (SEMM-Gln)

Semisolid MS + 2,4-D 18 µM + proline 50, 100, 150, 200 mM Maturation of somatic embryos; somatic embryo maturation

medium (SEMM-Pro)

Semisolid hormone free half strength MS Plantlet regeneration medium (PRM)


183

primary calli and frequency of formation of different

stages of somatic embryos by using phase contrast-

fluorescent microscope (Nikon, Japan): (a) 28 day old

primary calli derived from different explants types for

the determination of embryogenic/non-embryogenic

status and % embryogenic cells (EC); (b) 28 day old

secondary calli (end point of I subculture) developed

subsequent to stress-factor incubation; and (c) 28 day

old secondary calli (end point of II subculture)

developed in amino acids supplemented nutrient

medium. Total number of somatic embryos per 10 mg

dry weight of calli was determined by scoring the

total number of somatic embryos present in all stages

and by working out the dry weight of different calli.

In a population of somatic embryos, different stages

were scored and their percentage distribution

presented. The description of the calli as embryogenic

calli or non-embryogenic calli is based on the

presence of EC in the respective calli. % EC = No. of

globular cells/Total no. of cells in the EC × 100. A

hand lens with a magnification power of 3X was

employed for determining the callus emergence.

Callus emergence was monitored twice a day at

12 h interval.

Isolation and preparation of torpedo and cotyledonary stages

of somatic embryos from embryogenic calli

Torpedo and cotyledonary stage embryos were

separated from the callus mass by employing a

dissection microscope (providing 10X magnification)

and a fine needle and collected separately, free of

adjacent free cells, for the purpose of employing in

specified experiments. Globular and heart shaped

embryos could not be separated from the callus mass

by employing the dissection microscope based

methodology due to their very small and microscopic

size (ca. 250 µM).

Chlorophyll estimation

Total chlorophyll content was estimated according

to Arnon (1949)21

.

Dry weight determination

Weighed amounts of fresh weight of calli were dried

overnight (15 h) in a hot air oven at 110°C and the

dry weight determined. For determination of

somatic embryos frequency per 10 mg dry weight, total

number of somatic embryos present in all stages was

scored in pre-weighed amount of fresh weight of

embryogenic calli and by determining the dry weight of

the calli.

RNA isolation

Total RNA was isolated using the ‘Nucleospin

RNA plant kit’ (Macherey-Nagel, Germany) as per

the protocol given by the manufacturer. RNA was

quantified using absorbance at 260 nm and the quality

of the isolated RNA was assessed by loading onto a

1.4% formaldehyde agarose gel containing 5 mL of

10 X MOPS buffer. Total RNA was isolated from

different stages of the cultures as detailed in the

results related to expression analysis of SERK1 gene.

These included: (a) 28 day old nonembryogenic

primary calli raised from embryo axis explants in

MS + 2,4-D (9 uM); (b) 28 day old embryogenic

primary calli raised from embryo axis explants in

MS + 2,4-D (18 uM); (c) 28 day old secondary calli

(end point of I subculture) raised from 28 day

old nonembryogenic primary calli without PEG

incubation in MS+2,4,D (18 µM)-somatic

embryogenesis medium; (d) 28 day old secondary

calli (end of I subculture) raised from 28 day old

nonembrogenic primary calli with PEG incubation in

MS+2,4,D (18 µM)-somatic embryogenesis medium;

(e) 28 day old secondary calli (end of I sub-culture)

raised from 28 day old embrogenic primary calli

without PEG incubation MS+2,4,D (18 µM)-somatic

embryogenesis medium; (f) 28 day old secondary calli

(end point of I subculture) raised from 28 day old

embrogenic primary calli with PEG incubation in

MS+2,4,D (18 µM)-somatic embryogenesis medium

during secondary calli phase; (g) 28 day old

secondary calli (end point of I subculture) enriched

with globular stage somatic embryos raised from 28

day old embrogenic primary calli with 4% PEG (w/v)

incubation for 3 h in MS+2,4,D (18 µM)-somatic

embryogenesis medium during secondary callus

phase; (h) 28 d old secondary calli (end of I

subculture) enriched with heart stage somatic

embryos raised from 28 day old embryogenic primary

calli with 3% PEG (w/v) incubation for 6 h in

MS+2,4,D (18 µM)-somatic embryogenesis medium

during secondary callus phase; (i) isolated preparation

of torpedo stage somatic embryos from 28 day old

secondary calli (end point of I subculture) raised from

28 day old embryogenic primary calli raised from 28

day old embryogenic primary calli with 4% PEG

incubation for 6 h in MS+2,4,D (18 µM)-somatic

embryogenesis medium during secondary callus

phase; and (j) isolated preparation of cotyledonary

stage somatic embryos from 28 day old tertiary calli

(end point of II subculture) raised from 28 day old

embryogenic secondary calli, developed subsequent to 4%


184

PEG incubation for 6 h in MS+2,4,D (18 µM)-somatic

embryogenesis medium during secondary callus phase.

cDNA synthesis and semi-quantitative RT-PCR

cDNA was prepared from total RNA using

Transcriptor First Strand cDNA synthesis kit (Roche,

Switzerland). It was performed for 60 min at 50 and

85°C for 5 min. As the SERK1 gene sequence for V.

radiata was not available in the database, primers

were designed based on the conserved regions of the

nucleotide sequences of SERK1 genes of related

legume species available in the Geb Bank

[SERK1-mRNA sequences of Medicago truncatula

(AY162176.1), Glycine max (XM_003556137.1), Cicer

arietinum (XM_004496342.1) and A. thaliana

(FJ08762.1)]. The following set of primers was

designed (forward: 5′-TGGTGGCGTCGAAGGAAAC-

3′; reverse: 5′-CCTGGATTAACTGCTCTACCTCA-

3′). PCR (Eppendorf, Germany) was performed with

the following cycling conditions: 94°C for 45 s

(denaturation), 55°C for 45 s (annealing), 72°C for 1

min (extension) with one round of 34 cycles and the

final extension for 5 min at 72°C. The amplified

products were analyzed by electrophoresis on a 1.5%

agarose gel. Actin gene expression was used as an

endogenous control to ensure equal amounts of cDNA

in all lanes. PCR products were sequenced using

automated sequencer (ABI 3730xl Genetic). The

nucleotide sequences were submitted for similarity

search in the NCBI GenBank database using the online

BLAST programme (http://blast.ncbi.nlm.nih.gov)22

.

Data presentation

At least 72 explants cultured in 6 vessels with 12

explants per vessel were employed in each treatment.

For stress incubation experiments 6 culture vessels

containing primary calli developed from ca. 72 explants

were incubated with or without sorbitol/mannitol/PEG

incubation for each of the treatments. Each experiment

was repeated thrice. A complete randomized design was

used in all the experiments and analysis of variance and

mean separations were carried out using Duncan’s

Multiple Range Test23

. Significance was determined at

5% level. Data presented are the mean of 3 replicates

along with SD.

Results and Discussion

Induction of embryogenic calli and evaluation of embryogenic

potential

Induction of SE in cultures of legumes in general

and greengram in particular is known to be difficult

due to in vitro recalcitrance11,12,

. Establishment of

defined in vitro protocol for developing somatic

embryos needs to have experimental strategies that

have holistic approach encompassing different

parameters of the experimental system and to have no

inclusion of ill-defined culture conditions. Formation

of embryogenic calli consisting of potent cells for SE

has been shown to be a pre-requisite for developing

somatic embryos24

. In the present study, an elegant

approach to address and overcome the limiting factors

in greengram cultures for somatic embryo formation

has been attempted (Scheme 1).

In order to work out optimal conditions for the formation of embryogenic calli, a set of experiments employing different explant types prepared from source seedlings of various age and different concentrations and forms of auxins supplemented either separately or in combination with Kn was

carried out (Table 2). Callusing response was evaluated on the basis of a select set of parameters in order to assess the embryogenic potential of the cultures. Results showed that supplementation of auxin alone, either in the form of 2,4-D or IAA, resulted in the formation of embryogenic calli. 2,4-D

has been the preferred auxin in legume cultures for

Scheme 1 — Optimum protocol for high frequency development

of somatic embryos in cultures of green gram


185

the induction of SE11,16

. In order to evaluate the relative efficacy of 2,4-D and IAA for the formation

of embryogenic calli, a set of experiments was undertaken. Results showed that 2,4-D was more effective than IAA, at their equimolar concentrations, in the formation of embryogenic calli based on the % embryogenic cells in the calli. IAA when supplemented in place of 2,4-D brought about

qualitatively comparable callusing response in all the explant types employed although the quantum response was found to be lower. The 2,4-D being a synthetic auxin, is known to be metabolically more

stable due to resistance to auxin degrading enzymes and metabolic conversion to various storage forms of

auxins25

. Its supplementation in cultures is known to result in building up relatively higher intracellular concentration of this synthetic auxin leading to strong response as compared to native forms of auxin

26. There was no comparable trend in %

callusing response and formation of embryogenic

calli of the different explant types, such as, embryo axis, leaf and cotyledonary node under the different phytohormone regimes employed in the present study.

Table 2 — Effect of supplementation of 2, 4- D, IAA and Kn in various combinations and concentrations and evaluation of somatic

embryogenic potential of the primary calli based on a select set of morphogenetic parameters in cultures of greengram

Explants Phytohormone

supplements (µM)

Callusing response*

2,4-D IAA Kn Time of

appearance (d)

% Colour &

texture

Chlorophyll

content µg g fw-1 Embryogenic /non-

embryogenic

%

EC

Amount (mg

dry wt explant-1)

Embryo

axis

0 0 0 n.d. 0 n.d. 0 n.d. 0 0

9 0 0 8.5± 0.31c 64±2.2s GCF 138±4.2g NE 0 15±0.73de

18 0 0 4.0±0.16a 95±3.70a GYF 102±3.3d E 89a 25±1.18a

27 0 0 6.5±0.20b 72±2.8t GYF 116±3.4e E 61f 19±0.97e

36 0 0 7.5±0.29b 51±1.63q GYC 153±5.0f NE 0 9±0.44c

45 0 0 8.0±0.33c 48±1.49qp GF 191±6.1h NE 0 8±0.45c

0 18 0 6.5±0.27b 73±2.73w GYF 112±4.09e E 59f 18±0.90e

18 0 2.3 6.5±0.27b 55±1.70q GC 203±6.4i NE 0 14±0.68d

18 0 4.6 8.5±0.33c 64±2.31s GC 248±7.4a NE 0 18±0.87e

18 0 6.9 9.0±0.36c 52±1.56q GC 233±7.5k NE 0 13±0.62d

18 0 9.2 9.5±0.38c 50±1.84q GC 223±7.1j NE 0 12±0.6d

Leaf

0 0 0 n.d. 0 n.d. 0 n.d. 0 0

9 0 0 n.d. 0 n.d. 0 n.d. 0 0

18 0 0 15.5±0.56d 45±1.58p GWF 106±3.6de E 51g 10±0.5d

27 0 0 16.0± 0.67d 26±1.27i BWa 0 NE&S 0 7±0.35c

36 0 0 17.5± 0.72g 19±0.91f BWa 0 NE&S 0 5±0.24b

45 0 0 n.d. 0 n.d. 0 n.d. 0 0

0 18 0 16.0± 0.67d 28±1.35i GWF 109±4.1e E 33e 6±0.3b

18 0 2.3 17.5±0.72e 35±1.50l GWF 116±4.5ef E 15b 9±0.42c

18 0 4.6 18.5±0.76h 50±2.0rq GWF 121±4.9f E 26d 8±0.39c

18 0 6.9 18.0± 0.70h 31±1.39k GWF 110±4.05e E 20c 7±0.35c

18 0 9.2 18.5±0.83h 32±1.35kl GC 252±8.1a NE 0 7±0.35c

Cotyledonary

node

0 0 0 n.d. 0 n.d. 0 n.d. 0 0

9 0 0 n.d. 0 n.d. 0 n.d. 0 0

18 0 0 17.5±0.76e 18±0.70ef YF 20±0.82b NE 0 4±0.20b

27 0 0 16.5±0.69de 40±1.82m YF 23±0.94b E 17bc 8±0.4c

36 0 0 16.5±0.69de 29±1.35i BF 0 NE 0 3±0.15b

45 0 0 n.d 0 n.d. 0 n.d. 0 0

0 27 0 18.0±0.78k 25±1.05i YF 19±0.88b E 15b 7±0.35c

27 0 2.3 18.5±0.76k 29±1.19i YF 21±0.88b NE 0 3±0.14b

27 0 4.6 19.0±0.81k 11±0.52b YF 23±0.94b NE 0 5±0.25b

27 0 6.9 19.5±0.76k 15±0.67e YF 32±1.31l NE 0 4±0.04b

27 0 9.2 19.5±0.76k 8±0.37b YF 38±1.63l NE 0 5±0.23b

[*monitored on 28th day subsequent to explants culture; GCF- greenish creamy friable, GYF- greenish yellow friable, GYC- greenish

yellow compact, GC- greenish compact, GF- greenish friable, GWF- greenish white friable, BWa- brownish watery, YF-yellowish

friable, BF- brownish friable, NE- non embryogenic, E- embryogenic, n.d. – not detected]


186

There was significant difference in the

phytohormone optima that brought about

embryogenic response from the different explants

types. Embryo axis and leaf explants showed similar

optimum in 2,4-D concentration at 18 µM for the

formation of embryogenic calli (Fig. 1 F, I, J, L and M).

Based on the range of 2,4-D concentrations employed,

it could be inferred that there was an optimal range of

2,4-D concentration for induction of the desired in

vitro response viz., formation of embryogenic calli

from different explants types. 2,4-D supplementation

<18 µM and >27 µM did not result in the formation of

embryogenic calli in all the tested explant types. Even

though there was no formation of embryogenic calli

in the tested concentrations of Kn in combination with

optimal concentrations of 2,4-D in respect of embryo

axis and cotyledonary node, leaf explants exhibited

embryogenic response under comparable hormonal

Fig. 1 — In vitro responses in relation to SE in cultures of greengram [(A) yellowish friable calli from cotyledonary node explants raised

in MS + 2,4-D (18 µM); (B) Greenish creamy friable calli from embryo axis explants raised in MS + 2,4- D (9 µM); (C) Greenish yellow

friable calli from embryo axis explants raised in MS + 2,4- D (18 µM); (D) greenish yellow compact calli from embryo axis explants

raised in MS + 2,4-D (36 µM); (E) Greenish friable calli from embryo axis explants raised in MS + 2,4- D (18 µM)+ kn (2.3 µM); (F)

greenish compact calli from embryo axis explants raised in MS + 2,4-D (18 µM)+ kn (4.6 µM); (G) brownish friable calli from

cotyledonary node explants raised in MS + 2,4- D (36 µM); (H) brownish watery calli from leaf explants raised in MS + 2,4- D (27µM);

(I) Callus emergence in leaf explants cultures in MS + 2,4- D (18 µM)+ kn (4.6 µM) on 14d old primary cultures; (J) greenish white

friable leaf calli raised in MS + 2,4- D (18µM); (K) non-embryogenic cells in primary calli from embryo axis explants raised in MS +

2,4-D (9 µM); (L) embryogenic cells in primary calli from embryo axis explants raised in MS + 2,4-D (18 µM); (M) pro-embryogenic

mass in calli from embryo axis raised in MS + 2,4-D (18µM); (N) globular stage somatic embryo, (O) Heart stage somatic embryo in 28

day old secondary cultures; (P) torpedo stage somatic embryo in 28 day old secondary cultures; (Q) early cotyledonary stage somatic

embryo in 28 day old tertiary cultures subsequent to proline supplementation; (R) cotyledonary stage somatic embryo in 28 day old

tertiary cultures subsequent to proline supplementation; and (S) 23 day old plantlet raised from cotyledonary stage somatic embryo; bar in

(K, L, N-O):50 µm; bar in (P-R): 1 mm]


187

regime (Fig. 1K). Callusing response was found to be

high at relatively lower concentration of 2,4-D in

embryo axis and leaf explants as compared to

cotyledonary node explants.

Time of emergence of callus from different explant

types under the employed culture conditions displayed varied responses, such as early response (up to 8 days), late response (8.5-19.0 days) and no response. The % callusing response was found to vary among the different explants types employed in the present study under the imposed conditions. The order

of response was as follows: embryo axis>leaf >cotyledonary node. Even though the different combinations of 2,4-D and Kn resulted in higher callusing response, there was no formation of embryogenic callus except in leaf explants. In the present study, friable nature of the calli was found to

be associated with embryogenic attributes of the cultures. Also, the colour of the calli was found to be indicative of embryogenic nature. Calli which were greenish yellow friable, greenish creamy friable and yellowish friable were found to be embryogenic in contrast to compact calli along with greenish and

greenish yellow colouration (Fig. 1 A-C). Brownish watery calli was found to mark non-embryogenic calli by virtue of cessation of growth and attaining the senescence status (Fig. 1H). Chlorophyll content of the callus cultures marked the degree of differentiation in the cultures. Results showed that beyond a particular

stage of greening, the cultures were non-embryogenic (Fig.1 D and E). In the present study, brownish friable and brownish watery calli formed due to supplementation of 2,4-D at 36 µM to cotyledonary node explant cultures displayed onset of senescence resulting in the lysis of the cells (Fig. 1 G and H).

The 2,4-D concentration beyond a threshold level has been shown to inhibit cell growth and in vitro responses, possibly due to the operation of programmed cell death (PCD)

25-27. Formation of high

frequency embryogenic calli was found to be associated with early emergence of callus from

different explants types employed in the present study. Amount of calli produced explant

-1 on the basis

of dry weight had a positive correlation with the % callusing response observed in the different explants types. Also, there was relationship between amounts of calli produced explant

-1 and the formation of

embryogenic calli on the basis of % embryogenic cells. Evaluation of embryogenic competence of the calli on the basis of % embryogenic cells revealed a positive correlation with the growth of calli on the

basis of the amount of calli formed explant-1

. Based on the % embryogenic cells, there was an apparent inverse relationship between age of the seedlings from which explants were prepared and embryogenic nature of the different calli developed. Results

showed that all the explants formed embryogenic calli albeit at different combinations and concentrations of phytohormones. The order of response in producing embryogenic calli on the basis of % embryogenic cells from different explants was as follows: embryo axis>leaf>cotyledonary node. 2,4-D mediated

acquisition of embryogenic competence and formation of somatic embryos has been unequivocally shown in several systems, such as, A. thaliana and

Glycine max17,28

. In soybean, based on the steady state level of mRNA in immature cotyledon derived calli grown on 2,4-D supplemented medium, it has

been shown that the cells underwent two phases of development viz., dedifferentiation followed by redifferentiation into embryogenic cells

28. Comparably,

the results of the present study showed that under conditions of 2,4-D supplementation the calli derived from different explants types was found to have two

distinct phases including the later phase of formation of embryogenic cells. However, cell tracking experiments to work out the kinetics of the genesis of embryogenic cells in the callus mass was not undertaken in the present study for facilitating the analysis of the successive events that might occur

during dedifferentiation and redifferentiation.

Osmotic stress induced formation of somatic embryos in

embryogenic callus cultures

In continuation of our earlier work related to the

employment of osmolytes for the formation of

somatic embryos in pigeonpea in vitro and in order

to widen the application potentials of the previously

established protocol to yet another in vitro

recalcitrant legume species, calli of greengram were

subjected to incubation in different individually

supplemented osmolytes containing media (Table 3).

In order to work out ideal conditions for the

induction of SE and for high frequency somatic

embryo formation, optimization of the type and

concentration of osmolyte and duration of osmotic

stress incubation has been found to be essential17

. In

the present study, SE response due to stress factor

incubation was evaluated on the basis of total

number of somatic embryos, which included all the

different stages of somatic embryos that were

produced in the cultures.


188

Observations showed that there was no induction

of SE in either mannitol or sorbitol when

supplemented to the nutrient medium at 0.6 M

irrespective of the duration of incubation. Possibly,

this meant that the cellular osmolarity was iso-

osmolar with the external osmolarity contributed

mainly by the osmolytes and marginally by the

nutrients present in the medium. Accordingly, the

selected concentrations of the plasmolyzing osmolytes

viz., mannitol and sorbitol, supplemented individually

at 0.6, 0.7 and 0.8 M, pointed out the possibility of

being either iso-osmolar or hyper-osmolar and not

hypo-osmolar based on the induction of SE

subsequent to stress incubation. In the present study,

incubation duration of 6 h was found to be optimal for

the induction of SE by mannitol or sorbitol at a

Table 3 — Effect of incubation of embryogenic calli in osmolytes supplemented media on the formation of somatic

embryos in cultures of greengram

Osmolytes &

concentration

Incubation

Duration (h)

Somatic embryos

(10 mg dry wt of calli-1)

% Distribution of different stages of somatic embryos

Globular Heart Torpedo

No osmolyte

(control)

0 n.d. n.d. n.d. n.d.

3 n.d. n.d. n.d. n.d.

6 n.d. n.d. n.d. n.d.

9 n.d. n.d. n.d. n.d.

Sorbitol 0.6 M 0 n.d. n.d. n.d. n.d.

3 n.d. n.d. n.d. n.d.

6 n.d. n.d. n.d. n.d.

9 n.d. n.d. n.d. n.d.

Sorbitol 0.7 M

0 n.d. n.d. n.d. n.d.

3 30±1.17a 75±2.4a 18±0.86c 07±0.35b

6 52±1.69b 65±2.30b 22±0.90c 13±0.54b

9 32±1.31a 75±2.4a 17±0.85c 8±0.33b

Sorbitol 0.8 M

0 n.d. n.d. n.d. n.d.

3 09±0.40d 70±2.27a 27±1.12c 3±0.14b

6 27±1.10a 71±2.76a 22±1.03c 7±0.33b

9 n.d. n.d. n.d. n.d.

Mannitol 0.6 M

0 n.d. n.d. n.d. n.d.

3 n.d. n.d. n.d. n.d.

6 n.d. n.d. n.d. n.d.

9 n.d. n.d. n.d. n.d.

Mannitol 0.7 M

0 n.d. n.d. n.d. n.d.

3 36±1.44a 74±2.39a 18±0.89c 08±0.4b

6 61±1.97c 63±2.01b 20±0.92c 17±0.77d

9 33±1.35a 66±2.49b 24±1.12c 10±b

Mannitol 0.8 M

0 n.d. n.d. n.d. n.d.

3 14±0.51e 81±3.24ab 13±0.65a 06±0.47bc

6 37±1.44a 69±2.62a 17±0.78ac 14±b

9 n.d. n.d. n.d. n.d.

PEG 3%

0 n.d. n.d. n.d. n.d.

3 n.d. n.d. n.d. n.d.

6 13±0.58e 58±2.38c 38±1.67ae 4±0.66ab

9 06±0.28d 70±2.66a 30±1.41f 0

PEG 4%

0 n.d. n.d. n.d. n.d.

3 96±3.1g 72±2.88a 18±0.84c 10±0.47b

6 153±4.3f 47±2.35d 21±0.98c 32±1.33d

9 107±2.7h 67±2.88ab 19±0.78c 14±0.65bd

PEG 5%

0 n.d. n.d. n.d. n.d.

3 26±1.04ai 76±2.96a 20±0.83c 4±0.19b

6 72±2.44j 70±2.87a 21±1.02c 9±0.41b

[Twenty eight day old embryogenic primary calli were incubated in different osmolytes for indicated durations. Frequency of

occurrence of somatic embryos and % distribution of different stages of somatic embryos were determined in the secondary cultures

on 28th day subsequent to incubation in osmolytes supplemented media]


189

specific hyper-osmolar concentration. Sorbitol or

mannitol supplemented at 0.7 M resulted in the

optimal induction of SE and beyond 0.7 M, the

concentrations of the osmolytes were found to be

non-physiological resulting in irreversible cellular

damage possibly due to excessive exosmosis under

the employed duration of incubation.

In the present study, PEG incubation at 4% (w/v)

for 6 h resulted in the formation of somatic embryos

at the highest frequency as compared to the other

employed osmolytes and incubation durations. Stress

due to different osmolytes and duration of incubation

resulted in the formation of somatic embryos at

different stages, such as globular, heart and torpedo at

varying frequency in the cultures (Fig. 1 N-P).

However, the cultures did not produce any significant

number of cotyledonary stage embryos under those

conditions. It could be inferred that there could be

stage specific requirements of phytohormones, growth

factors and other factors including compatible

osmotica for the maturation of the somatic embryos in

cultures. Karami et al.29

found that once the process

of SE commenced in cultures it was an auto-

regulatory process having no other specific

requirement of growth factors or only minimal

requirement of them for the formation of matured

somatic embryos. Thus, it could be perceived that

induction and progression of SE culminating in the

formation of cotyledonary stage embryo is not a one

trigger process in greengram cultures. Development

of somatic embryos in the cultures of the present

study was found to be nonsynchronized and the

presence of different stage of somatic embryos could

be observed in all the embryogenic cultures which

were developed subsequent to stress factor incubation.

Dehydration stress to cultured tissues or cells induced

by externally supplemented osmolytes at

hyperosmolarity is known to mimic the conditions

that exist in the embryo sac during zygotic

embryogenesis17

. It could be visualized that at iso-

osmolar concentration of the osmolytes, the calli cells

do not undergo osmotic stress. In contrast, hypo-

osmolar or hyper-osmolar concentration of the

osmolytes would exert osmotic stress on the calli

cells. Results related to stress induced formation of

somatic embryos in cultures of A. thaliana at hypo-

osmolar concentration of osmolytes indicated lack of

attainment of SE response under those conditions17

.

Hence, the present study did not focus on hypo-

osmolar stress incubation in relation to induction of

SE. Possibly, hypo-osmotic stress would result in cell

burst due to endosmosis, thus inhibiting growth and

development in cultures.

In the present study, cells with embryogenic

competence, characterized by a typical microscopically

observable cellular morphology, not subjected to

osmotic stress incubation did not develop somatic

embryo when primary cultures were sub-cultured on

2,4-D supplemented medium. Also, non-embryogenic

cells did not undergo SE in 2,4-D supplemented

medium subsequent to PEG incubation. Effects of

PEG in different embryogenic systems of carrot,

wheat, white spruce and A. thaliana pointed out the

significant influence of dehydration stress due to this

non-plasmolyzing osmolyte in the induction of

SE15,16,18

. Interestingly, Ikeda-Iwai et al.17

reported

that in A. thaliana dehydration stress created by mere

placing of the explants on sterilized dry filter paper

lead to the formation of somatic embryos on

subsequent transfer to 2,4-D containing medium.

As compared to stress inducible occurrence of SE in

A. thaliana, where explants prepared from seedlings

and mature plants were employed for stress

incubation, the present study utilized embryogenic

calli. Comparable to the A. thaliana system, culture of

calli subsequent to stress incubation in 2,4-D

supplemented medium sustained embryogenic

competence and resulted in the development of

somatic embryos. When stress incubated embryogenic

calli were transferred to 2,4-D lacking medium there

was no progression in the SE pathway and the

cultures altogether ceased to grow any further and

underwent senescence (data not shown).

Osmotic/dehydration stress imposed at an optimal

concentration of stress factor and duration of

incubation might induce two processes, viz. (a)

expression of stress inducible genes and b) conferring

embryogenic competence involving dedifferentiation

and redifferentiation of the somatic cells27,30

.

Subsequent to stress factor incubation and on transfer

to stress factor omitted medium containing 2,4-D,

adaptability of the cells might take place to regain

cellular homeostasis including the original osmolarity.

Subsequent to this phase of stress adaptability by the

cells in the 2,4-D supplemented medium, there could

be sustainability of the acquired embryogenic

competence. It is known that there are comparable

cellular and molecular events associated with

hyperosmotic-stress adaptability and SE18,28

. The

2,4-D supplementation to cultures subsequent to stress


190

factor incubation of calli could confer sustenance of

the acquired embryogenic competence of the cells and

regulate their further development in the SE pathway.

However, the exact mechanism of cellular responses

to osmotic stress and associated occurrence of SE in

cultures has not been understood well18,25

.

In order to sustain the osmotic stress induced

cellular and molecular changes, other than the

cytosolic osmotic potential which gets reverted,

subsequent to stress factor incubation the calli were

grown on 2,4-D containing medium where 2,4-D

itself could act as a stress17

. Importance of duration of

stress incubation in relation to optimal response

leading to the expression of stress inducible genes and

also parallel conferring of competence for SE and

development of somatic embryos have been

highlighted in several studies1. Under supraoptimal

conditions of stress incubation, the cells would

undergo irreversible damage resulting in the loss of

viability. It has been shown in the embryogenic callus

cultures of soybean which were developed from

immature cotyledons on 2,4-D containing medium,

that the globular stage embryos were formed in four

weeks28

. In the present study, 4 wk old primary callus

cultures, which were not subjected to stress factor

incubation, produced calli enriched with embryogenic

cells (EC) under optimal culture conditions. In the

light of findings related to somatic embryogenesis in

legume cultures and based on the observations of

the present study, it could be inferred that 2,4-D is

required continuously for implementation of the

somatic embryogenesis programme with an

intervening short period of stress factor incubation in

2, 4-D lacking medium. Cultured tissues, especially

long term cultures, are known to undergo nuclear

changes in relation to mutation, endoduplication of

chromosomes and ploidy level. In the present study,

in order to control in vitro caused nuclear changes of

the calli very young age primary calli were employed

for inducing SE9.

Proline mediated maturation of early stage somatic embryos

to cotyledonary stage

Results showed that subsequent to stress factor

incubation and on transfer to 2,4-D supplemented

medium, the callus cultures showed lack of

development of cotyledonary stage somatic embryos.

In order to convert the early stage somatic embryos to

maturation, amino acid supplements, such as

tryptophan, glutamine and proline, were

supplemented individually to the medium.

Observations showed that proline supplementation

alone supported the formation of cotyledonary stage

somatic embryos (Table 4 and Fig. 1 Q and R).

Supplementation of reduced form of nitrogen in the

form of either casein hydrolysate or individual amino

acids has been shown to influence the maturation of

early stage somatic embryos31,32

. Proline is known to be

a compatible osmoticum involved in the maintenance

of cellular osmolarity under conditions of changing

cellular osmotic potential. When the somatic embryos

undergo maturation it is possible that the cells of the

embryos undergo gradual and steady increase in the

cellular osmotic potential. Accumulation of proline

might contribute to the build-up of the required

osmotic potential of cytosol during the maturation

phase that occurs in the cotyledonary stage embryos.

Germination of plantlets from matured cotyledonary stage

somatic embryos

In the present study, cotyledonary stage somatic

embryos were subsequently developed into plantlets

in hormone-free half strength semisolid MS medium

at a frequency of ca. 81%. The in vitro developed

plantlets were subsequently grown in garden soil at a

survival rate of ca. 100% (Fig. 1S). A total of 54

plantlets were developed in garden soil.

Table 4 — Effect of of amino acid supplements on the

maturation / formation of cotyledonary stage somatic

embryos in cultures of greengram.

Amino acid

supplements

Concentration

(mM)

% of cotyledonary

embryos

L- Try

0 2±0.23g

50 4±0.21g

100 7±0.33h

150 9±0.43f

200 3±0.14g

L- Gln

0 4±0.36g

50 7±0.34h

100 10±0.47f

150 15±0.70e

200 5±0.25g

L- Pro

0 3±0.54g

50 25±1.12c

100 32±1.39b

150 53±2.01a

200 21±0.95d

[Twenty eight day old embryogenic secondary callus cultures

containing early stage somatic embryos, such as, globular, heart

and torpedo were transferred to amino acids supplemented media

for the maturation to cotyledonary stage. Occurrence of somatic

embryos at the end of the tertiary phase of callus cultures was

evaluated. Individual aminoacids were supplemented to semisolid

MS+ 2,4-D (18µM) at indicated concentrations]


191

Expression analysis of SERK1 gene in embryogenic calli and

in somatic embryos at different stages

In order to work out a molecular marker for SE and

to analyze the timing of expression of the selected

marker gene during SE, a set of experiments

involving the profiling of the expression of SERK1

was carried out. The SERK1 gene of V. radiata

was amplified using gene specific primers (forward:

5′-TGGTGGCGTCGAAGGAAAC-3′; reverse:

5′-CTGGATTAACTGCTCTACCTCA- 3′). The 1305

bp partial cDNA of SERK1 gene amplified from

V. radiata (vrSERK1) showed 93% homology

with SERK1 of G. max and 90% homology with

SERK1 of M. truncatula. Semi-Quantitative Reverse

Transcription - PCR analysis showed the expression

of SERK1 in the embryogenic primary calli and

globular and heart stage of somatic embryos of the

callus cultures which were developed subsequent to

PEG incubation of the embryogenic primary calli

(Fig. 2). Nonembryogenic primary calli which were

analysed either prior to PEG incubation or subsequent

to PEG incubation and after 28 days did not show the

expression of SERK1 even when cultured on the 2,

4-D containing medium during both the phases.

Results showed the expression of the gene in the non-

synchronized embryogenic cultures that contained

enriched population of heart stage somatic embryos

along with globular stage somatic embryos. In this

case, contribution of the globular stage somatic

embryos to the detected SERK1 expression could not

be ruled out. It is pertinent to mention that globular

and heart stage somatic embryos could not be

separately isolated from the embryogenic calli in the

present study. Results showed unequivocally that

SERK1 expression was not detected in the

homogenous preparation of torpedo and cotyledonary

stage of somatic embryos. Interestingly, embryogenic

calli consisting of embryogenic cells which showed

expression of SERK1 during the primary callus phase

did not exhibit the expression of SERK1 in the

absence of PEG incubation when transferred to 2,4-D

containing medium subsequent to primary callus

phase. Possibly, embryogenic competence marked by

the expression of SERK1 in the embryogenic calli at

the end of the primary callus phase was short lived

and it did not continue beyond the primary callus

phase. However, more work is needed to evaluate the

embryogenesis potential of the callus cultures in

relation to culture age.

Expression of several genes, such as, SERK1,

LEC1, LEC2, FUS3/ABI3 and BBM has been shown

to be associated with the onset of SE and subsequent

development of somatic embryos29,33

. In particular,

expression of SERK1 was shown to be associated

with embryogenic competence and somatic to

embryogenic transition in cultures of diverse plant

species, such as D. carota, A. thaliana,

M. truncatula, G. max, Helianthus annus, Dactylis

glomereta and Oryza sativa29

. SERK1 gene has

also been shown to be a marker of early somatic

embryogenesis in D. carota, D. glomereta and

A. thaliana34,35

. A few reports provided evidence

for the expression of SERK1 associated with

stages other than the early stages of SE and also

in tissues and physiological processes not related

to SE, such as, primary meristems of root and shoot,

junction between one type of tissue or organ

and another, vascular tissues and procambial

cells, flowers, host-defense response against fungal

infection, regulation of male sporogenesis for

tapetum development and microspore maturation30,36

.

Results of the present study showed that the

expression of SERK1 gene was markedly absent in

various other stages of the cultures, such as, non-

embryogenic cells in primary calli, and later stages

of SE, such as, torpedo and cotyledonary stages of

somatic embryos. The present study provides

evidence for the first time related to the presence and

expression of SERK1 gene in embryogenic cells and

globular stage somatic embryos in greengram.

SERK1 gene is known to be a membrane bound

protein that encodes for leucine rich repeat receptor

like kinases (LRR-RLKs) family of plant protein

kinases and involved in 2,4-D and brassinosteroid

signaling27,29

. SERK has been shown to act as

receptor for brassinosteroids but not for

2,4-D in Arabidopsis thaliana37

.

Fig. 2 — Semi-quantitative Reverse Transcription PCR - based

expression analysis of SERK1 gene in cultures of greengram [Lane

1, non-embryogenic primary calli; Lane 2, embrogenic primary

calli; Lane 3, non-embryogenic primary calli without PEG

incubation; Lane 4, non-embrogenic primary calli with PEG

incubation; Lane 5, embryogenic primary calli without PEG

incubation; Lane 6, embrogenic primary calli with PEG incubation;

Lane 7, globular stage somatic embryos; Lane 8, Heart stage

somatic embryos; Lane 9, torpedo stage somatic embryos; and Lane

10, cotyledonary stage somatic embryos]


192

Results of the present study provided evidence

possibly for the first time regarding upregulation of

SERK1 gene in embryogenic cultures of greengram

and also its expression specifically associated with the

early stages of SE. In addition, molecular data related

to the expression of SERK1 obtained in the present

study provided additional evidence for the genesis of

somatic embryos in these cultures. It is pertinent to

mention that somatic embryos have been converted to

synseeds for long term storage in cultures of Digitalis

davisiana38

Also, an elegant microdroplet cell

cultures have been found to be suitable for single

cell derived microcalli and somatic embryos in

cultures of pigeonpea39

.

In conclusion, it could be stated that the present

study contributes to the establishment of a high

frequency plantlet regeneration protocol via SE by

employing embryogenic callus cultures derived from

embryo axis and incubation in PEG. Also, the study

contributed to pinpoint the expression of SERK1 gene

only during early stages of SE. Based on the results of

the present study related to SERK1 expression, it

could be inferred that there are two distinct phases in

the SE pathway consisting of the development of

embryogenic cells independent of dehydration stress

and the subsequent phase of SE having dependency

on stress incubation.

Acknowledgement

This work was supported by DBT, Government of

India (BT/PR9030/AGR/02/401/2007) to KM.

Additional support to the work by UGC-NRCBS

[F.10/2008(BSR)], DBT-IPLS (BT/R14553/INF/22/

124/2010), DST-FIST (SR/FST/LS11-013/2009)

DST-PURSE and UGC – CAS – Phase III, School of

Biological Sciences, Madurai Kamaraj University, is

gratefully acknowledged. VS (DST-INSPIRE) and

MG (DBT-IPLS) thank for fellowships.

References 1 Chugh A & Khurana PJ, Gene expression during somatic

embryogenesis-recent advances. Curr Sci, 83 (2002) 715.

2 Tiwari AK & Shivhare AK, Pulses in India Retrospect &

Prospects, (Publication No.: DPD/Pub.1/Vol. 2/2016,

Directorate of Pulses Development, Ministry of Agri. &

Farmers Welfare (DAC&FW), Govt. of India, Bhopal,

India), 2016.

3 Heuzé V, Tran G, Bastianelli D, Lebas F, Mung bean (Vigna

radiata). Feedipedia, (a programme by INRA, CIRAD, AFZ

and FAO), 2015. http://www.feedipedia.org/node/235

4 Anonymous, Mung Beans - Nutrition & Its Big Benefits, In:

Dr Axe’s Food is Medicine. https://draxe.com/mung-beans-

nutrition/

5 Kapravelou G, Martínez R, Nebot E, López-Jurado M,

Aranda P, Arrebola F, Cantarero S, Galisteo M & Porres JM,

The Combined Intervention with Germinated Vigna radiata

and Aerobic Interval Training Protocol Is an Effective

Strategy for the Treatment of Non-Alcoholic Fatty Liver

Disease (NAFLD) and Other Alterations Related to the

Metabolic Syndrome in Zucker Rats. Nutrients, 9 (7) (2017).

pii: E774. doi: 10.3390/nu9070774.

6 Kumar P, Chand R, Singh V & Pal C, In vitro screening of

mungbean to cercosporin, a photoactivated toxin. Indian J

Exp Biol, 55 (2017) 113.

7 Singh AK, Singh SS, Prakash V, Kumar S and Dwivedi SK,

Pulses Production in India: Present Status, Bottleneck and

Way Forward. J Agrisearch, 2(2) (2015) 75.

8 Zavattieri MA, Induction of somatic embryogenesis as an

example of stress-related plant reactions. Electr J Biotechnol,

13 (2010) 1.

9 Chandra A & Pental D, Regeneration and genetic

transformation of grain legumes: An overview. Curr Sci,

84 (2003) 381.

10 Somers DA, Samac DA & Olhoft PM, Recent advances in

legume transformation. Plant Physiol, 131 (2003) 892.

11 Graham PH & Vance CP, Legumes: Importance and

constraints to greater use. Plant Physiol, 131 (2003) 872.

12 Kaviraj CP, Kiran G, Venugopal RB, Kavi Kishor PB &

Srinath rao, Somatic embryogenesis and plant regeneration

from cotyledonary explants of green gram [Vigna radiata

(L.) Wilezek.] – a recalcitrant grain legume. In Vitro Cell

Dev Biol Plant, 42 (2006) 134.

13 Siva Kumar P, Gnanam R, Ramakrishnan R & Manickam A,

Somatic embryogenesis and regeneration of Vigna radiata L.

Biol Plant, 54 (2010) 245.

14 Prathibha Devi P, Radha L, Sitamahalakshmi D, Syamala S

& Manoj Kumar, Plant regeneration via somatic

embryogenesis in mung bean [Vigna radiata (L.) Wilczek].

Scientia Horticulturae, 99 (2004) 1.

15 Harada H, Kiyosue T, Kamada H & Kobayashi T,

Stress-induced carrot somatic embryogenesis and its

application to synthetic seeds, In: The impact of

Biotechnology in Agriculture, (RS Sangwan and RS

Sangwang Norreel (Eds), Kluwer Academic Publishers,

The Neetherlands), (1990) 129.

16 Taras PP, Prinsen E, Ayaydin F, Miskolczi P, Potters G,

Asard H, Harry A, Onckelen V, Dudits D & Feher A, The

role of auxin, pH and stress in the activation of embryogenic

cell division in leaf protoplast-derived cells of alfalfa. Plant

Physiol, 129 (2002) 1807.

17 Ikeda-Iwai M, Umehara M, Satoh S & Kamada H,

Stress-induced somatic embryogenesis in vegetative tissues

of Arabidopsis thaliana. Plant J, 34 (2003) 107.

18 Stasolla C, Zyl LV, Egertsdotter U, Craig D, Liu W &

Sederoff, RR, The effects of polyethylene glycol on gene

expression of developing white spruce somatic embryos.

Plant Physiol, 131 (2003) 49.

19 Viji M, Maheswari P, Karuppanapandian T & Manoharan K,

Effect of polyethylene glycol and mannitol on somatic

embryogenesis of pigeonpea, Cajanus cajan (L.) Millsp.

African J Biotech, 11 (2012) 10340.

20 Murashige T & Skoog K, A revised medium for rapid growth

and bioassays with tobacco tissue cultures. Physiol Plant, 15

(1962) 473.


193

21 Arnon DI, Copper enzymes in isolated chloroplasts,

polyphenoxidase in Beta vulgaris. Plant Physiol,

24 (1949) 1.

22 Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z,

Miller W & Lipman DJ Gapped BLAST and PSI-BLAST: a

new generation of protein database search programs. Nucleic

Acids Res, 25 (1997) 3389.

23 Duncan DB, Multiple range and multiple F test. Biometrics,

11 (1955) 42.

24 Girija S, Ganapathi A & Ananthakrishnan G, Somatic

embryogenesis in Vigna radiata (L.) Wilczek. Indian J Exp

Biol, 38 (2000) 1241.

25 Raghavan V, Role of 2,4-Dichlorophenoxy acetic acid

(2,4-D) in somatic embryogenesis on cultured zygotic

embryos of Arabidopsis: cell expansion, cell cycling, and

morphogenesis during continuous exposure of embryos to

2,4-D. Am J Bot, 91 (2004) 1743.

26 Ribnicky DM, Ilic N, Cohen JD & Cooke TJ, The effects of

exogenous auxins on endogenous indole-3-acetic acid

metabolism, The implications for carrot somatic

embryogenesis. Plant Physiol, 112 (1996) 549.

27 Smertenko A & Bozhkov PV, Somatic embryogenesis: life

and death processes during apical–basal patterning. J Exp

Bot, 65 (2014) 1343.

28 Thibaud NF, Shealy RT, Khanna A & Vodkin LO,

Clustering of microarray data reveals transcript patterns

associated with somatic embryogenesis in soybean (Glycine

max L.). Plant Physiol, 132 (2003) 118.

29 Karami O, Aghavaisi B & Mahmoudi Pour A, Molecular

aspects of somatic-to-embryogenic transition in plants.

J Chem Biol, 2 (2009) 177.

30 Karami O & Saidi A, The molecular basis for stress-induced

acquisition of somatic embryogenesis. Mol Biol Rep, 37

(2010) 2493.

31 Constance N, Chowdhry AK, Tyagi N & Maheshwari SC, Effect

of L-proline and L-tryptophan on somatic embryogenesis and

plantlet regeneration of rice (Oryza sativa L. cv. Pusa 169).

Plant Cell Tissue Org Cul, 32 (1993) 357.

32 Rengel Z & Jelaska S, The effect of L-proline on somatic

embryogenesis in long-term callus culture of hordeum

vulgar. Acta Bot Croat, 45 (1986) 71.

33 Becraft PW, Receptor kinase signaling in plant development.

Annu Rev Cell Dev Biol, 18 (2002) 163.

34 Salaj J, von Recklinghausen IR, Hecht V, de Vries SC,

Schel JHN & van Lammeren AM, AtSERK1 expression

precedes and coincides with early somatic embryogenesis in

Arabidopsis thaliana. Plant Physiol Biochem, 46 (2008) 709.

35 Thakare D, Tang W, Hill K & Perry SE, The MADS-domain

transcription regulator AGAMOUS-LIKE15 promotes

somatic embryo development in Arabidopsis and soybean.

Plant Physiol, 146 (2008) 1663.

36 Nolan KE, Kurdyukov S & Rose RJ, Expression of the

SOMATIC EMBRYOGENESIS RECEPTOR LIKE

KINASE1 (SERK1) gene is associated with developmental

change in the life cycle of the model legume Medicago

truncatula. J Exp Bot, 60(6) (2009) 1759.

37 Albrecht C, Russinova E, Kemmerling B, Kwaaitaal M & de

Vries SC, Arabidopsis somatic embryogenesis receptor kinase

proteins serve brassinosteroid-dependent and independent

signaling pathways. Plant Physiol. 148 (2008) 611.

38 Verma SK, Sahin G & Gurel E, Somatic embryogenesis,

pigment acculumation, and synthetic seed production

in Digitalis davisiana Heywood (Alanya foxglove). Indian J

Exp Biol, 54 (2016) 245.

39 Kumar NU, Gnanaraj M, Sindhujaa V, Viji M & Manoharan

K, A microdroplet cell culture based high frequency somatic

embryogenesis system in pigeonpea, Cajanus cajan (L.)

Millsp. Indian J Exp Biol, 53(4) (2015) 600.

induction of high frequency somatic embryogenesis and...

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